Module 3, Lab 10 - Protein quantitationBradford dtermination of GFP

Today we did a protein quantitation using BioRad's Quick Start™ Bradford Protein Assay, a method in which a dye reagent is used (Bradford reagent, based on Brilliant Blue G-250) to bind to proteins (causing the dye reagent to change from a reddish-brownish color to blue) and measure its absorbance. The more concentrated the protein it binds, the darker the blue resultant color, and the greater the absorbance at 595 nm.

Two relative standard proteins are used, bovine serum albumin (BSA) and bovine gamma-globulin (BGG), to generate absorbance vs. protein concentration curves and then interpolate the absorbance of problem samples (mostly with GFP) to estimate their concentration. The problem samples were obtained from the Hydrophobic Interaction Chromatography (HIC).

This method is applied when researchers in proteomics discover a new protein and are trying to gather information about it. In our case, we "discovered" GFP, although we wouldn't have a name yet, had it been a truly newly discovered protein.

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Module 3, Lab 09 - Hydrophobic Interaction Chromatography (HIC) of GFP

Friday, October 21, 2010

Today we performed a Hydrophobic Interaction Chromatography (HIC) to separate the green fluorescent protein (GFP), produced in our bacterial cultures, from other proteins commonly found in bacteria.

A sample of bacteria was concentrated and then resuspended in a solution in which they were lysed. The high salt solution, containing all the proteins found in the bacteria, was then passed through a hydrophobic interaction column where molecules of GFP bound to the hydrophobic beads. The high salt solution increased the hydrophobicity of GFP by further exposing its hydrophobic amino acid residues.

A series of washes with buffers of decreasing salinity allows proteins with various levels of hydrophobicity to gradually unbind from the beads and be collected in a test tube. By switching collection tubes each time a buffer is added, different proteins can be collected. One of them was GFP and the tube in which it was collected should glow.

Diagram of Hydrophobic Interaction Chromatography (HIC)

GFP molecules are represented by black triangles

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Module 2, Lab 07 - Sequencing reactions of GAPC gene

After the cloning process of the GAPC gene from Arabidopsis, and extracting the plasmid DNA (to isolate the pJet1.2 plasmid) we did a RED to confirm the success of the ligation.

Using the samples that had the GAPC gene insert we mixed purified plasmid DNA with forward and reverse sequencing primers (pJET SEQ F and pJET SEQ R), and put them in a 96-well plate. The plate will be shipped to the DOE Joint Genome Institute (JGI) to be sequenced as part of their Sequencing Training Program (STR). The results should be in in two weeks, ready to be used in the bioinformatics labs

We will discuss the DNA sequencing technique most commonly used: Dye-terminator sequencing, a modification of Sanger's chain termination sequencing protocol, which allowed the automation of the DNA sequencing process.

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Guest lecture by Dr. Renee Reijo-PeraEarly human embryo development and associated gene expression

We had the privilege of having Dr. Renee Reijo-Pera, the director of the Human Embryonic Stem Cell Research Center at Stanford University, as a guest lecturer in our class. She shared with us her lab's findings in recent years on stem cell research and early human embryo development.

Some of the main topics in her lecture included...

• Maternal vs. embryonic gene expression - Stages at which maternal mRNAs are active, and then degraded, and at which embryonic mRNAs are synthesized
• Dynamics of cell division between fertilization and blastocyst stage
• Prediction, at day 2 of development, of which embryos are viable (will successfully reach blastocyst stage) - Development of an algorithm to make an objective prediction
• Things we do not know about human embryo development and how stem cell research can help
• How embryo images were obtained and made into movies to allow analysis of developmental process
• Analysis of gene expression - analysis of mRNA from 96 selected genes, extracted from a single cell
• How the development process is correlated with patterns of gene expression

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Lecture, chapter 11 - RNA processing+ info about guest lecturer, Dr. Renee Reijo-Pera

Today we finished chapter 11 on RNA processing. We focused mainly on alternative splicing and how it produces different mRNA molecules by transcribing the same gene.

We also discussed processes like base modification, base substitution, RNA editing, and RNA degradation.

After finishing the chapter we discussed students' impressions on Dr. Reijo-Pera's Keiser lecture yesterday evening and expectations for her talk in our class tomorrow...!!! (expectations from the talk itself and about students' interaction with Dr. Reijo-Pera)

Tomorrow:

Dr. Renee Reijo-Pera, from Stanford University, will give a lecture on human preimplantation development and gene expression and pathways during the first few days of development. We will be joined by students in Dr. Aulthouse's Developmental Anatomy class, and potentially Dr. Walden's CLS program so the room will be packed. The talk will be as exciting as the Keiser lecture and having two-three classes in the audience will make the discussion more interesting and lively...!

Students should be ready to ask questions to, and engage in a discussion with, Dr. Reijo-Pera. Please check the following links:

• The Reijo-Pera lab
• People at the Reijo-Pera lab
• Dr. Reijo-Pera's publications
• Dr. Reijo-Pera's contact info

It will be an exciting day. Take advantage of it!

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Upcoming lecturer: Dr. Renee Reijo-Pera

On Wednesday we will have the honor of having Dr. Renne Reijo-Pera as a guest lecturer in our class. Dr. Reijo Pera is visiting ONU as the Distinguished Keiser Lecturer of 2010.

Her lecture is titled Human Health, Development and Stem Cells, and it will be delivered on Monday, October 18, at 7:00 pm at the Freed Center

In our class Dr. Reijo-Pera will be talking about human preimplantation development and gene expression and pathways during the first few days of development. This topic is highly appropriate for our class and it will provide you with the opportunity to interact in an intimate setting with a world-class researcher. Take advantage of such opportunity!

Dr. Reijo-Pera is the Director of the Human Embryonic Stem Cell Research and Education Center at Stanford University. For more information see her lab web page, which includes information about her and her group, and her publications.

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Module 2, Lab 06 - Cloning - RED of the plasmid DNApurifications

(entry in progress)

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Module 2, Lab 06 - Cloning (GAPC gene from Arabidopsis and pJet1.2 plasmid)

Today we used the E. coli cultures you inoculated yesterday to do a small-scale plasmid DNA extraction ('miniprep') using Promega's Wizard® Plus SV Minipreps DNA Purification System.

Here's a recap of what we have done so far in the last couple of exercises:

Lab 5 - Nested PCR of the GAPC gene from Arabidopsis

Lab 6 - Cloning

• Ligation of PCR amplified GAPC gene onto the pJet1.2 plasmid
• Genetic transformation of E. coli with the pJet1.2 plasmid
• Cloning of genetically transformed E. coli
• Minipreps (purification of pJet1.2 plasmid)

Next, a restriction enzyme digestion to confirm successful plasmid DNA purification (lab 6) and setting sequencing reactions (lab 7)

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Module 3, Lab 08 - Genetic transformation of E. coli with the pGLO plasmid

Jen was kind enough to prepare new LB agar plates and redo the transformation of E. coli with the pGLO plasmid. Then she plated the transformed bacteria on the plates she prepared, some of which had ampicillin, arabinose, or both.

[Fantastic] Results are shown in the pictures below:

(entry in progress)

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Lecture, chapter 11 - RNA processing

tRNA processing

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Today we started chapter 11, on RNA processing, the fifth "stop" in our roadmap.

We mentioned the most basic processes through which RNA is modified (base modification, cleavage, and splicing) and described the more complex processes that are specific to eukaryotic RNA (5' capping and 3' polyadenylation).

We discussed in more detail the steps through which introns are spliced out the pre-mRNA molecule, and introduced the concept of alternative splicing.

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Lecture, chapter 10 - Gene regulation in eukaryotes

Today we covered chapter 10, on gene regulation in eukaryotes.

We highlighted the differences in between the regulatory processes in prokaryotes and the more complex processes in eukaryotes, mainly the facts that in eukaryotes many transcription factors are needed to aid transcription and that DNA is many times condensed in heterochromatin, and therefore unavailable for RNA polymerase and other proteins.

Among some of the processes that affect gene expression we discussed histone acetylation, a mechanism through which heterochromatin is relaxed into euchromatin, making DNA available for DNA binding proteins, and methylation, a mechanism through which genes can be repressed or silenced, in some cases because it promotes de-acetylation of histones and DNA condensation into heterochromatin.

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Module 2, Lab 06 - Ligation and transformation (GAPC gene from Arabidopsis and pJet1.2 plasmid)

Today we purified the PCR products from the nested PCR lab (GAPC gene from Arabidopsis) and used them to genetically transform E. coli.

The lab was divided in three main steps

• Ligation (of GAPC gene on to the pJet1.2 plasmid)
• Preparation of competent cells
• Genetic transformation of E. coli

We spent most of the lab manipulating bacteria to make them competent (i.e. get them ready to uptake extracellular DNA). Once this was achieved, the GAPC gene from Arabidopsis, obtained via nested PCR, was ligated to the pJet1.2 plasmid.
The plasmid was then used to genetically transform E. coli, which were spread on LB agar/Amp/IPTG plates and incubated.

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Lecture, chapter 09 - Gene regulation in prokaryotes

Today we lectured for a little over one hour, as a replacement for the lab we could not do, because of not having transformed bacteria to "play" with.

We finished chapter 9 on gene regulation in prokaryotes. We discussed the concepts of positive and negative regulation, including the role of (specific) activators and repressors, and global regulators. We studied how the Lac operon works in E. coli, and set the stage for starting with gene regulation in eukaryotes.

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Lecture, chapter 09 - Gene regulation in prokaryotes

We started the fourth stop in our "road map" (how genes are regulated).

We introduced chapter 09, on gene regulation, by highlighting the importance of the process for both eukaryotic and prokaryotic cells. We also mentioned the different steps during the process of information transfer at which regulation can take place, transcription being the most common one.

Note: Because the results of the transformation lab were negative (E. coli genetic transformation failed) we will lecture tomorrow.

• Section 01: 8:45 am
• Section 02: 10:00 am
• (Any one who wants to attend the opposite section is welcome)

Information on how to obtain the results from the transformation lab will be posted on an update in that exercise's blog entry.

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Lecture, chapter 7 - Protein structure and function

We finished chapter 7...

We discussed the features that allow certain proteins to bind to DNA and also what of their most common structural motifs are. We closed by briefly describing what protein denaturation is, how it differs from degradation, and what are the most common denaturing agents.

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Lecture, chapter 7 - Protein structure and function

Today we covered most of chapter 7, on protein structure and function.

We discussed the properties a protein has given the characteristics amino acids provide to the whole structure and how they actually affect protein function. We also listed different protein categories according to different functions they may have.

Tomorrow we will talk about how proteins can recognize DNA sequences to bind to them.

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Module 3, Lab 08 - Genetic transformation of bacteria with the pGLO plasmid

Aequorea victoria, original source of the green fluorescent protein (GFP)

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Today we used the pGLO plasmid to genetically transform Escherichia coli.

pGLO is a plasmid that has been engineered to contain the Green Fluorescent Protein (GFP) gene, originally isolated from the jelly Aequorea victoria. GFP produces a green fluorescence when excited by blue or UV light.

In order to make the GFP gene a functional one it has been engineered so the sugar arabinose triggers the production of the protein. The genes in the arabinose operon (araB, araA, and araD) have been replaced by the GFP gene. Such genes encode proteins that break down arabinose when it is present in the environment, so they are expressed only if this is the case. The regulatory sequence has been left intact, so in the engineered operon the presence of arabinose turns on the GFP gene and, therefore, GFP is produced.

Another feature of the pGLO plasmid is the presence of the beta-lactamase gene, which provides resistance against the antibiotic ampicillin.

The bacteria were transformed through the heat shock technique, and then plated on LB agar plates containing:
Just LB (lysogeny broth)
LB and ampicillin
LB, ampicillin and arabinose

Plates are being incubated for 24 hours at 37ºC.

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Module 2, Lab 05 - GAPDH Nested PCR

Today we ran a gel electrophoresis to confirm the results of the second round of PCR (using the nested primers).

Ideally, we would have purified the Arabidopsis PCR product (GAPC gene; in fact, section 1 did follow the process), but all PCRs failed. WHY?

Here's what I think happened:

When I retrieved the tubes from the thermocycler they had a very small volume of liquid at the bottom of the tube. Most of the volume (pretty much all the water, not just the water you added) was condensed at the top of the tube. That means that all the reagents were desiccated and molecules couldn't interact with each other. Result: no reaction whatsoever.

Probable cause: A glitch in the thermocycler. Most thermocyclers today have heated lids, to prevent condensation of water at the top of the tube. There is evaporation, but by preventing condensation water is always being recirculated in between its liquid and gas states and there is always enough liquid water to keep the PCR going. If the lid doesn't heat up during the process, then most of the water evaporates, condensates at the top of the tube and the reaction is ruined.

I have no idea of why the lid wouldn't heat up, since it is an automatic process every time you run a program.

Solution: I am running the nested PCRs again. The first round is in the thermocycler as I type and I triple-checked to make sure the lid was hot. I will run the second round and a gel to make sure that we have product (GAPC gene). If the reaction works, in week 5, before the ligation and transformation exercise, you will have to purify the PCR product before proceeding.

Lesson: Learn how to deal with frustration. In molecular biology many things can go wrong when following a protocol and you must keep on going. If you ever become part of a research lab you will find out, first hand, that nothing is as perfect as it looks in the published literature. Today you had a little taste of it.

We must shake it off and do it again. In this case I have to do it again (but if there are any volunteers for setting up the second round of PCRs, let me know)

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Results of make up PCRs

The following gels show the results of the nested PCR (click on the pic to see a full size image):

Lanes 1 and 2 on both gels are the positive and negative controls, of the first round PCR on the left gel and of the nested PCR (2nd round) on the right. The box on the left gel shows a faint band, which resulted from leaking when I was loading the positive control in the adjacent well.

Lanes 3 and 4 on the left gel show products of the first round of PCR, and all other lanes in both gels show products of the second round of PCR.

I have saved the products of the nested PCRs for you to purify this week and go on with lab 6.

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Exam 1

Statistics of the exam:

(click on pic for full size image)

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Lecture, chapter 6 - Transcription

Today we finished the chapter on transcription.

We discussed the process in which RNA polymerase binds to the promoter in prokaryotes, generates de RNA transcript, and reaches the terminator to end transcription (Rho-independent termination and Rho-dependent termination).

We then discussed eukaryotic transcription. The promoter is more complex (it has an initiator box, a TATA box, and upstream elements), there are three different RNA polymerases that transcribe nuclear genes (mitochondrial and chloroplast genes use other polymerases), and there are proteins, called transcription factors (general and specific), that aid RNA polymerases in the transcription of genes. Some proteins may bind to enhancer regions, upstream from the promoter, to aid in the process.

Watch the video that we saw in class (embedded action disabled in Youtube)

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